Recombinant rabies virus genome plasmid for high-sensitivity expression of red fluorescent protein, recombinant rabies virus as well as preparation method and application of recombinant rabies virus genome plasmid

Abstract

The invention discloses a recombinant rabies virus genome plasmid for high-sensitivity expression of red fluorescent protein, a recombinant rabies virus as well as a preparation method and application of the recombinant rabies virus. The preparation method specifically comprises the following steps: (1) constructing the recombinant rabies virus genome plasmid for high-sensitivity expression of red fluorescent protein as shown in SEQ ID NO.2; (2) preparing a recombinant rabies virus for expressing the red fluorescent protein with high sensitivity; and (3) application of the recombinant rabies virus for high-sensitivity expression of the red fluorescent protein in neural circuit tracing. The recombinant rabies virus capable of highly sensitively expressing the red fluorescent protein can efficiently mark an upstream neural network in a retrograde manner, so that the problem of low efficiency of expressing red fluorescence in the existing method is solved; the method has wide application value and market prospect in the aspects of neural circuit structure and function analysis, neuron sparse marking, vaccine and drug research and development, disease model establishment, neural network loss and reconstruction detection, virus replication or pathogenic mechanism research and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant rabies virus genome plasmid for highly sensitively expressing red fluorescent protein, a recombinant rabies virus, and a preparation method and application thereof. Background Art

[0002] Neural circuits are extremely complex, and their abnormalities often lead to the occurrence of brain diseases such as Alzheimer's disease and Parkinson's disease. There is still a lack of effective treatment methods. The main reason includes that the mutation mechanism of the neural network is still unclear. The retrograde tracing tool virus based on rabies virus can be used to analyze the connection and function of the neural network between the center and the center, and between the periphery and the center, and has great application value in the field of neuroscience. Compared with its retrograde transsynaptic ability, the retrograde tracing tool virus based on CVS-N2c-ΔG has significantly enhanced retrograde transsynaptic ability and less toxicity to neurons. It can express functional probes such as calcium ion probes and optogenetic probes for the analysis of functional circuits, and has been widely used in the study of neural circuit structure and function. However, the existing CVS-N2c-ΔG still has problems such as low efficiency in expressing red fluorescence, and it takes 7 days to achieve high expression of fluorescence. Therefore, it is of great significance to develop a recombinant rabies virus that expresses red fluorescent protein with high sensitivity. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a recombinant rabies virus genome plasmid that expresses red fluorescent protein with high sensitivity, a recombinant rabies virus, and a preparation method and application thereof, aiming to solve the problem of low efficiency of existing recombinant rabies virus in expressing red fluorescence.

[0004] The technical solution of the present invention is as follows:

[0005] In a first aspect, a recombinant rabies virus genome plasmid that expresses red fluorescent protein with high sensitivity is provided, and the nucleotide sequence of the recombinant rabies virus genome plasmid is shown in SEQ ID NO.2.

[0006] In a second aspect, a method for preparing the recombinant rabies virus genome plasmid as described in the first aspect is provided, the preparation method comprising the following steps:

[0007] (1) synthesizing a DNA fragment expressing red fluorescent protein, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0008] (2) The DNA fragment and CVS-N2c-ΔG-tdTomato plasmid were double-digested with SmaI and NheI respectively and then ligated to obtain a recombinant rabies virus genome plasmid with a nucleotide sequence as shown in SEQ ID NO.2.

[0009] In a third aspect, a recombinant rabies virus expressing a red fluorescent protein with high sensitivity is provided. The recombinant rabies virus is obtained by co-transfecting cells with the recombinant rabies virus genome plasmid and the backbone plasmid as described in the first aspect.

[0010] In a fourth aspect, a method for preparing the recombinant rabies virus according to the third aspect is provided, the method comprising the following steps:

[0011] (1) co-transfecting the recombinant rabies virus genome plasmid and the backbone plasmid as described in claim 1 into B7GG cells for virus rescue and amplification to obtain CVS-N2c-ΔG-3×Opti-mScarlet-I virus;

[0012] (2) BHK-N2cG cells were infected with CVS-N2c-ΔG-3×Opti-mScarlet-I virus to obtain N2cG-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus.

[0013] In a preferred technical solution, the backbone plasmid includes pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G.

[0014] According to a preferred technical solution, the mass ratio of the recombinant rabies virus genome plasmid, pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G is 4:2:1:1:1.

[0015] A preferred technical solution is that the method for preparing BHK-N2cG cells comprises the steps of:

[0016] (1) connecting the H2B-GFP-P2A-N2cG fragment with the nucleotide sequence as shown in SEQ ID NO.3 to a lentiviral expression vector to obtain a lentiviral core plasmid;

[0017] (2) The lentiviral core plasmid and the lentiviral backbone plasmid were co-transfected into HEK-293T cells, and the lentivirus was collected and infected into BHK-21 cells to obtain BHK-N2cG cells that stably expressed nuclear localized green fluorescence and N2cG protein.

[0018] In a preferred technical solution, the lentiviral expression vector is FUGW.

[0019] In a preferred technical solution, the lentiviral backbone plasmid includes pMDLg / pRRE, pRSV-Rev and pMD2.G.

[0020] In a fifth aspect, a use of the recombinant rabies virus genome plasmid as described in the first aspect or the recombinant rabies virus as described in the third aspect in neural circuit tracing is provided.

[0021] A preferred technical solution is to use the recombinant rabies virus genome plasmid or the recombinant rabies virus in the preparation of drugs for neural circuit tracing.

[0022] A preferred technical solution is to apply the recombinant rabies virus genome plasmid or the recombinant rabies virus to the analysis of neural circuit structure and function, sparse neuron labeling, vaccine and drug development, disease model establishment, detection of neural network loss and reconstruction, and study of viral replication or pathogenic mechanism.

[0023] Beneficial effects: The present invention provides a highly sensitive recombinant rabies virus genome plasmid expressing red fluorescent protein, a recombinant rabies virus, and a preparation method and application thereof, specifically comprising: (1) constructing a highly sensitive recombinant rabies virus genome plasmid expressing red fluorescent protein as shown in SEQ ID NO.2; (2) preparing a highly sensitive recombinant rabies virus expressing red fluorescent protein; (3) using the highly sensitive recombinant rabies virus expressing red fluorescent protein in neural circuit tracing. The highly sensitive recombinant rabies virus expressing red fluorescent protein of the present invention can efficiently retrogradely mark the upstream neural network, solving the problem of low efficiency of expressing red fluorescence in the existing method, and has broad application value and market prospects in the analysis of neural circuit structure and function, sparse labeling of neurons, vaccine and drug development, establishment of disease models, detection of neural network loss and reconstruction, virus replication or pathogenic mechanism research, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the map of CVS-N2c-ΔG-3×Opti-mScarlet-I plasmid.

[0025] Figure 2 Red fluorescence imaging of HEK-293T cells infected with N2cG-coated CVS-N2c-ΔG-3×Opti-mScarlet-I virus and N2cG-coated CVS-N2c-ΔG-tdTomato virus for 24 hours and 48 hours, respectively.

[0026] Figure 3 This is the red fluorescent signal of the injection site and upstream brain area 3 days after infection with N2cG-encapsulated CVS-N2c-ΔG-tdTomato virus.

[0027] Figure 4 This is the red fluorescence signal of the injection site and upstream brain area 3 days after infection with N2cG-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus. DETAILED DESCRIPTION

[0028] The present invention provides a recombinant rabies virus genome plasmid expressing red fluorescent protein with high sensitivity, a recombinant rabies virus, and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0029] The embodiment of the present invention provides a recombinant rabies virus genome plasmid that expresses red fluorescent protein with high sensitivity, and the nucleotide sequence thereof is shown in SEQ ID NO.2.

[0030] Specifically, the existing recombinant rabies virus genome plasmid CVS-N2c-ΔG-tdTomato has a low efficiency in expressing red fluorescent protein. Based on this, the inventor optimized the mScarlet-I sequence to obtain the Opti-mScarlet-I sequence, and then connected three Opti-mScarlet-I sequences through the QGGSGG sequence, and introduced restriction site genes at both ends to obtain the SmaI-Opti-mScarlet-I-QGGSGG-Opti-mScarlet-I-QGGSGG-Opti-mScarlet-I-NheI sequence that expresses red fluorescent protein with high sensitivity, and its nucleotide sequence is shown in SEQ ID NO.1. Further, the sequence expressing red fluorescent protein with high sensitivity is inserted into the CVS-N2c-ΔG-tdTomato plasmid, and finally a recombinant rabies virus genome plasmid expressing red fluorescent protein with high sensitivity is obtained, as shown in SEQ ID NO.2.

[0031] The present invention provides a method for preparing the recombinant rabies virus genome plasmid as described above, comprising the following steps:

[0032] (1) synthesizing a DNA fragment expressing red fluorescent protein, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0033] (2) The DNA fragment and CVS-N2c-ΔG-tdTomato plasmid were double-digested with SmaI and NheI respectively and then ligated to obtain a recombinant rabies virus genome plasmid with a nucleotide sequence as shown in SEQ ID NO.2.

[0034] The embodiment of the present invention provides a recombinant rabies virus expressing red fluorescent protein with high sensitivity, which is obtained by co-transfecting cells with the recombinant rabies virus genome plasmid and the backbone plasmid as described above.

[0035] The present invention provides a method for preparing the recombinant rabies virus as described above, comprising the following steps:

[0036] (1) co-transfecting the recombinant rabies virus genome plasmid and the backbone plasmid as described in claim 1 into B7GG cells for virus rescue and amplification to obtain CVS-N2c-ΔG-3×Opti-mScarlet-I virus;

[0037] (2) BHK-N2cG cells were infected with CVS-N2c-ΔG-3×Opti-mScarlet-I virus to obtain N2cG-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus.

[0038] In one embodiment, the backbone plasmid includes pCAG-B19P (addgene, catalog number: #59925), pCAG-B19N (addgene, catalog number: #59924), pCAG-B19L (addgene, catalog number: #59922) and pCAG-B19G (addgene, catalog number: #59921), but is not limited thereto.

[0039] In one embodiment, the mass ratio of the recombinant rabies virus genome plasmid, pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G is 4:2:1:1:1, but is not limited thereto.

[0040] In one embodiment, the method for preparing BHK-N2cG cells comprises the steps of:

[0041] (1) connecting the H2B-GFP-P2A-N2cG fragment with the nucleotide sequence as shown in SEQ ID NO.3 to a lentiviral expression vector to obtain a lentiviral core plasmid;

[0042] (2) The lentiviral core plasmid and the lentiviral backbone plasmid were co-transfected into HEK-293T cells, and the lentivirus was collected and infected into BHK-21 cells to obtain BHK-N2cG cells that stably expressed nuclear localized green fluorescence and N2cG protein.

[0043] In one embodiment, the lentiviral expression vector is FUGW (addgene, catalog number: #14883), but is not limited thereto.

[0044] In one embodiment, the lentiviral backbone plasmid includes pMDLg / pRRE (addgene, catalog number: #12251), pRSV-Rev (addgene, catalog number: #12253) and pMD2.G (addgene, catalog number: #12259).

[0045] An embodiment of the present invention provides a use of the above-mentioned recombinant rabies virus genome plasmid or the above-mentioned recombinant rabies virus in neural circuit tracing.

[0046] In one embodiment, the recombinant rabies virus genome plasmid or the recombinant rabies virus is used to prepare a drug for neural circuit tracing.

[0047] In one embodiment, the recombinant rabies virus genome plasmid or the recombinant rabies virus is applied to the analysis of neural circuit structure and function, sparse neuron labeling, vaccine and drug development, disease model establishment, detection of neural network loss and reconstruction, and study of viral replication or pathogenic mechanism.

[0048] The present invention will be further described below by means of specific examples.

[0049] Example 1

[0050] This embodiment provides a recombinant rabies virus genome plasmid and a recombinant rabies virus prepared therefrom, and the specific steps are as follows:

[0051] 1. Construction of recombinant rabies virus genome plasmid loaded with red fluorescent protein

[0052] In order to obtain a recombinant rabies virus genome plasmid carrying red fluorescent protein, the mScarlet-I sequence was optimized to obtain the Opti-mScarlet-I sequence, and restriction site genes were introduced at both ends to synthesize a DNA fragment expressing red fluorescent protein, named SmaI-Opti-mScarlet-I-QGGSGG-Opti-mScarlet-I-QGGSGG-Opti-mScarlet-I-NheI sequence, and its nucleotide sequence is shown in SEQ ID NO.1. The DNA fragment expressing red fluorescent protein was double-digested with SmaI and NheI and then connected and inserted into the CVS-N2c-ΔG-tdTomato plasmid (addgene, catalog number: #73462) that had been double-digested with the same restriction enzymes to obtain a recombinant rabies virus genome plasmid named CVS-N2c-ΔG-3×Opti-mScarlet-I plasmid. The map of the CVS-N2c-ΔG-3×mScarletI-I plasmid is shown in Figure 1 The full-length sequence is shown in SEQ ID NO. 2. All primers used in PCR, gene synthesis and sequencing of the present invention were completed by Beijing Qingke Biotechnology Co., Ltd.

[0053] 2. Establishment of BHK-N2cG stable cell line

[0054] The H2B-GFP-P2A-N2cG fragment with the nucleotide sequence shown in SEQ ID NO. 3 was synthesized. H2B-GFP-P2A-N2cG was connected into the FUGW vector (addgene, catalog number: #14883) linearized by BamHI and EcoRI by homologous recombination using the OneStep Cloning Kit (Vazyme) to obtain a lentiviral core plasmid named FUGW-H2B-GFP-P2A-N2cG. The correct lentiviral core plasmid FUGW-H2B-GFP-P2A-N2cG verified by sequencing was co-transfected with the lentiviral backbone plasmids pMDLg / pRRE (addgene, catalog number: #12251), pRSV-Rev (addgene, catalog number: #12253), and pMD2.G (addgene, catalog number: #12259) into HEK-293T cells. The fluorescence was observed after 24h and 48h, and the supernatant was collected at 48h and 72h, and filtered with a 0.22μm filter membrane to obtain the FUGW-H2B-GFP-P2A-N2cG lentivirus. The collected FUGW-H2B-GFP-P2A-N2cG lentivirus was tested for infection titer and then infected with BHK-21 cells (according to the infection multiplicity MOI = 5), and then placed in a cell culture incubator at 35°C and 3% (v / v) CO2 for 24 hours, and then fresh medium was replaced. After 72 hours, the cell state and fluorescence ratio were observed and passaged. When the fluorescence ratio reached more than 90%, the cells were preserved after 5 passages. The obtained stable cell line with nuclear localized green fluorescence was named BHK-N2cG.

[0055] 3. Establishment of a recombinant rabies virus expressing red fluorescence with high sensitivity

[0056] B7GG cells were cultured in DMEM medium containing 10% FBS and 1% double antibody (provided by SALK Institute for Biological Studies, USA). Before virus rescue, 12-well plates were plated with a cell density of 40%-50%. 1 mL of DMEM medium containing 10% FBS and no double antibody was added to each well and cultured overnight in a 37°C and 5% (v / v) CO2 incubator. After 16 hours, the medium in the well was removed and 1 mL of reduced serum medium Opti-MEM (Gibco) was added, and the cells were cultured in a 37°C and 5% CO2 incubator for use. CVS-N2c-ΔG-3×Opti-mScarlet-I plasmid (recombinant rabies virus genome plasmid) and backbone plasmids pCAG-B19P (addgene, catalog number: #59925), pCAG-B19N (addgene, catalog number: #59924), pCAG-B19L (addgene, catalog number: #59922), and pCAG-B19G (addgene, catalog number: #59921) were added to a 1.5 mL centrifuge tube pre-filled with 200 μL of opti-MEM medium at a mass ratio of 4:2:1:1:1, and vortexed on a vortex shaker to mix. After mixing, FUGENE 6. 18 μL of transfection reagent was vortexed and mixed, then placed at room temperature for 15 minutes and added to the corresponding wells of the 12-well plate. After continued cultivation for 6 hours in a 35°C and 3% (v / v) CO2 incubator, the culture medium in the well was replaced with DMEM medium containing 2% FBS and continued to be cultured in a 35°C and 3% CO2 incubator. After 72-120 hours of transfection, the cells were observed under an inverted fluorescence microscope. The appearance of red fluorescence signal indicated that the CVS-N2c-ΔG-3×Opti-mScarlet-I virus was successfully rescued.

[0057] The cells were then passaged and amplified for 10-14 days under culture conditions of 35°C and 3% (v / v) CO2 to obtain the B19G-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus. The supernatant of the B19G-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus was added to BHK-N2cG cells and amplified for 5-7 days to obtain the N2cG-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus. For details on the steps of virus amplification, concentration, purification and preservation, please refer to the literature (NeuralRegen Res.2023;18(8):1827-1833). The titer of the final N2cG-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus was 2×10 8 IU / mL.

[0058] In order to verify that the efficiency of the CVS-N2c-ΔG-3×Opti-mScarlet-I virus of the present invention in expressing red fluorescence is significantly better than that of the existing system, 1 μL of N2cG-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus and 1 μL of N2cG-encapsulated CVS-N2c-ΔG-tdTomato virus (Neural Regen Res. 2023; 18(8): 1827-1833) were taken to infect HEK-293T cells respectively (N2cG-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus was the experimental group, and N2cG-encapsulated CVS-N2c-ΔG-tdTomato virus was the control group, with a titer of 2×10 8 IU / mL), and then placed in a 37°C and 5% (v / v) CO2 incubator for further culture. Then, fluorescence imaging was performed using the same exposure parameters. The results are shown in Figure 2 shown. Figure 2 In the figure, the left picture shows the red fluorescence imaging of HEK-293T cells infected with CVS-N2c-ΔG-tdTomato virus wrapped with N2cG for 24 hours and 48 hours, with an exposure time of 500ms; the right picture shows the red fluorescence imaging of HEK-293T cells infected with CVS-N2c-ΔG-3×Opti-mScarlet-I virus wrapped with N2cG for 24 hours and 48 hours, with an exposure time of 500ms; under the same exposure conditions, the fluorescence brightness of the right picture is significantly better than that of the left picture. Figure 2 It can be seen that the efficiency of expressing red fluorescent protein by the CVS-N2c-ΔG-3×Opti-mScarlet-I virus prepared by the present invention is significantly higher than that of the CVS-N2c-ΔG-tdTomato virus (Neural Regen Res. 2023; 18(8): 1827-1833).

[0059] IV. In vivo application of recombinant rabies virus expressing red fluorescent protein with high sensitivity

[0060] The prepared N2cG-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus (100 nL / mouse, titer 2×10 8IU / ml) and CVS-N2c-ΔG-tdTomato virus wrapped with the same titer and dose of N2cG were respectively injected into the primary motor cortex (M1) area of ​​8-10 week old C57BL / 6 mice (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) by stereotaxic brain localization. Three days after infection, 0.5mL 1% (w / v) sodium pentobarbital-0.9% (w / v) sodium chloride solution was injected intraperitoneally into the mice for anesthesia, and the brain tissue was excised by cardiac perfusion using phosphate buffer saline (PBS) and 4% paraformaldehyde solution (PFA). The mouse brain tissue was fixed with PFA solution overnight and then dehydrated with 30% (w / v) sucrose-PBS solution. The dehydrated brain tissue was fully embedded with tissue embedding agent and cut into 40μm thick brain slices with a freezing slicer. The slices were imaged with an Olympus VS120 slide scanning microscope. The results are shown in Figure 2. Figure 3 and Figure 4 The results of in vivo detection showed that the CVS-N2c-ΔG-3×Opti-mScarlet-I virus wrapped with N2cG could efficiently mark the neurons in the injection site M1 and its upstream brain regions after 3 days of infection, and the fluorescence brightness expressed in living neurons was significantly better than that of the CVS-N2c-ΔG-tdTomato virus wrapped with N2cG (control group).

[0061] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

[0062] The nucleotide sequence of the present invention is as follows:

[0063] SEQ ID NO.1:

[0064]

[0065] SEQ ID NO.2:

[0066]

[0067] SEQ ID NO.3:

[0068]

Claims

1. A recombinant rabies virus genome plasmid expressing red fluorescent protein with high sensitivity, characterized in that: The nucleotide sequence of the recombinant rabies virus genome plasmid is shown in SEQ ID NO.

2.

2. A method for preparing a recombinant rabies virus genome plasmid as claimed in claim 1, characterized in that: The preparation method comprises the following steps: (1) synthesizing a DNA fragment expressing red fluorescent protein, the nucleotide sequence of which is shown in SEQ ID NO.1; (2) The DNA fragment and CVS-N2c-ΔG-tdTomato plasmid were double-digested with SmaI and NheI respectively and then ligated to obtain a recombinant rabies virus genome plasmid with a nucleotide sequence as shown in SEQ ID NO.

2.

3. A recombinant rabies virus expressing red fluorescent protein with high sensitivity, characterized in that: The recombinant rabies virus is obtained by co-transfecting cells with the recombinant rabies virus genome plasmid and the backbone plasmid as claimed in claim 1.

4. A method for preparing a recombinant rabies virus as claimed in claim 3, characterized in that: The preparation method comprises the following steps: (1) co-transfecting the recombinant rabies virus genome plasmid and the backbone plasmid as described in claim 1 into B7GG cells for virus rescue and amplification to obtain CVS-N2c-ΔG-3×Opti-mScarlet-I virus; (2) BHK-N2cG cells were infected with CVS-N2c-ΔG-3×Opti-mScarlet-I virus to obtain N2cG-encapsulated CVS-N2c-ΔG-3×Opti-mScarlet-I virus.

5. The method for preparing the recombinant rabies virus according to claim 4, characterized in that: The backbone plasmids include pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G.

6. The method for preparing the recombinant rabies virus according to claim 5, characterized in that: The mass ratio of the recombinant rabies virus genome plasmid, pCAG-B19P, pCAG-B19N, pCAG-B19L and pCAG-B19G is 4:2:1:1:

1.

7. The method for preparing the recombinant rabies virus according to claim 4, characterized in that: The method for preparing the BHK-N2cG cells comprises the steps of: (1) connecting the H2B-GFP-P2A-N2cG fragment with the nucleotide sequence as shown in SEQ ID NO.3 to a lentiviral expression vector to obtain a lentiviral core plasmid; (2) The lentiviral core plasmid and the lentiviral backbone plasmid were co-transfected into HEK-293T cells, and the lentivirus was collected and infected into BHK-21 cells to obtain BHK-N2cG cells that stably expressed nuclear localized green fluorescence and N2cG protein.

8. The method for preparing the recombinant rabies virus according to claim 7, characterized in that: The lentiviral expression vector is FUGW.

9. The method for preparing the recombinant rabies virus according to claim 7, characterized in that: The lentiviral backbone plasmids include pMDLg / pRRE, pRSV-Rev and pMD2.G.

10. Use of the recombinant rabies virus genome plasmid as claimed in claim 1 or the recombinant rabies virus as claimed in claim 3 in neural circuit tracing.

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